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Originally published In Press as doi:10.1074/jbc.M105324200 on October 31, 2001

J. Biol. Chem., Vol. 277, Issue 1, 395-401, January 4, 2002
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Mutation of Residue 33 of Human Equilibrative Nucleoside Transporters 1 and 2 Alters Sensitivity to Inhibition of Transport by Dilazep and Dipyridamole*

Frank Visserabcd, Mark F. Vickersabc, Amy M. L. Ngbe, Stephen A. Baldwinf, James D. Youngbeg, and Carol E. Cassabchij

From the a Canadian Institutes of Health Research Group in the Molecular Biology of Membrane Proteins and b Membrane Transport Research Group, Departments of h Biochemistry, c Oncology, and e Physiology, University of Alberta and the i Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada and the f School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom

Human equilibrative nucleoside transporters (hENT) 1 and 2 differ in that hENT1 is inhibited by nanomolar concentrations of dipyridamole and dilazep, whereas hENT2 is 2 and 3 orders of magnitude less sensitive, respectively. When a yeast expression plasmid containing the hENT1 cDNA was randomly mutated and screened by phenotypic complementation in Saccharomyces cerevisiae to identify mutants with reduced sensitivity to dilazep, clones with a point mutation that converted Met33 to Ile (hENT1-M33I) were obtained. Characterization of the mutant protein in S. cerevisiae and Xenopus laevis oocytes revealed that the mutant had less than one-tenth the sensitivity to dilazep and dipyridamole than wild type hENT1, with no change in nitrobenzylmercaptopurine ribonucleoside (NBMPR) sensitivity or apparent uridine affinity. To determine whether the reciprocal mutation in hENT2 (Ile33 to Met) also altered sensitivity to dilazep and dipyridamole, hENT2-I33M was created by site-directed mutagenesis. Although the resulting mutant (hENT2-I33M) displayed >10-fold higher dilazep and dipyridamole sensitivity and >8-fold higher uridine affinity compared with wild type hENT2, it retained insensitivity to NBMPR. These data established that mutation of residue 33 (Met versus Ile) of hENT1 and hENT2 altered the dilazep and dipyridamole sensitivities in both proteins, suggesting that a common region of inhibitor interaction has been identified.

* This work was supported by a Canadian Cancer Society grant from the National Cancer Institute of Canada (to C. E. C.), grants from the Canadian Institutes of Health Research (to C. E. C. and J. D. Y.) and the Wellcome Trust and Medical Research Council (to S. A. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

d Supported by studentships from the Alberta Heritage Foundation for Medical Research and the Endowed Ph.D. Studentship in Oncology.

g Heritage Scientist of the Alberta Heritage Foundation for Medical Research.

j Canada Research Chair in Oncology. To whom correspondence should be addressed: Dept. of Oncology, Cross Cancer Inst., 11560 University Ave., Edmonton, AB T6G 1Z2, Canada. Tel.: 780-432-8320; Fax: 780-432-8425; E-mail: carol.cass@cancerboard.ab.ca.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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